A HPLC-based glycoanalytical protocol allows the use of natural O-glycans derived from glycoproteins as substrates for glycosidase discovery from microbial culture.

ABSTRACT

Many disorders are characterised by changes in O-glycosylation, but analysis of O-glycosylation has been limited by the availability of specific endo- and exo-glycosidases. As a result chemical methods are employed. However, these may give rise to glycan degradation, so therefore novel O-glycosidases are needed. Artificial substrates do not always identify every glycosidase activity present in an extract. To overcome this, an HPLC-based protocol for glycosidase identification from microbial culture was developed using natural O-glycans and O-glycosylated glycoproteins (porcine stomach mucin and fetuin) as substrates. O-glycans were released by ammonia-based ß-elimination for use as substrates, and the bacterial culture supernatants were subjected to ultrafiltration to separate the proteins from glycans and low molecular size molecules. Two bacterial cultures, the psychrotroph Arthrobacter C1-1 and a Corynebacterium isolate, were examined as potential sources of novel glycosidases. Arthrobacter C1-1 culture contained a ß-galactosidase and N-acetyl-ß-glucosaminidase when assayed using 4-methylumbelliferyl substrates, but when defucosylated O-glycans from porcine stomach mucin were used as substrate, the extract did not cleave ß-linked galactose or N-acetylglucosamine. Sialidase activity was identified in Corynebacterium culture supernatant, which hydrolysed sialic acid from fetuin glycans. When both culture supernatants were assayed using the glycoproteins as substrate, neither contained endoglycosidase activity. This method may be applied to investigate a microbial or other extract for glycosidase activity, and has potential for scale-up on high-throughput platforms.